Lentiviruses, such as HIV-1, HIV-2, and SIV, infect not only activated/dividing CD4+ T cells but also non-dividing cells such as macrophages, microglia (brain macrophages), and resting CD4+ T cells. The HIV-1 infected myeloid cells such as macrophages and microglia serve as long-living viral reservoirs that persistently produce viral progenies. Importantly, while HIV-1 rapidly replicates in activated CD4+ T cells, the viral production from HIV-1 infected macrophages is kinetically suppressed. More importantly, unlike HIV-1, HIV-2 and many SIV strains (i.e. SIVmn) are able to replicates rapidly even in macrophages, and this fast replication kinetics of HIV-2/SIVsm in macrophages is induced by a virally encoded protein, Vpx. Cellular dNTPs are substrates of not only cellular DNA polymerases for chromosomal DNA replication but also DNA polymerases of various pathogens including HIV-1 reverse transcriptase (RT). In 2004, we reported that nondividing human primary macrophages contain extremely low dNTP pools (20-40nM), while activated CD4+ T cells harbor much higher dNTP concentrations (2-5M). We demonstrated that the low dNTP abundance is responsible for the suppressed replication kinetics of HIV-1 observed in macrophages, proposing that the limited dNTP availability is a nondividing cell specific metabolic restriction factor against HIV-1. In 2011, a new macrophage specific anti-HIV factor, SAMHD1, was identified, and this protein is dNTPase that hydrolyzes dNTP to dN and triphosphate. Vpx counteracts SAMHD1 and promotes HIV-2 replication in macrophages. Our work revealed that SAMHD1 is responsible for the low dNTP concentration of macrophages, and Vpx promotes HIV-2 replication kinetics in macrophages by elevating cellular dNTP levels and accelerating the reverse transcription step. In the previous funding period, we validated that Vpx reduces 1) the efficacy of nucleos(t)ide RT inhibitors in macrophages by elevating cellular dNTPs levels, and 2) HIV-1 recombination efficiency by limiting the kinetic-delay induced RT pausing and strand transfer in macrophages. In this new funding period, we propose to reveal two new additional steps of HIV-1 life cycle that are affected by the SAMHD1-mediated cellular dNTP starvation, 1) HIV-1 DNA gap filling step of viral integration, and 2) endogenous reverse transcription of HIV-1 in macrophages, because these two steps also consume and require dNTPs. In addition, we propose to solve the structure of the full-length SAMHD1 protein, which is not currently available, by employing innovative approaches. Finally, we will also investigate the structure and function of the SAMHD1-like protein of C. elegans, which controls the host developmental processes as observed in the human SAMHD1 genetic defects (Aicardi Goutieres Syndrome). This renewal application will provide innovative insights that can specifically target HIV-1 infection to nondividing myeloid cells that serve as long living viral reservoirs and contribute to HIV-1 persistence.